Heat shock protein gp96 is an important endoplasmic reticulum (ER) chaperone for peptides and proteins. A fraction of the gp96-associated peptides are trimmed and loaded onto MHC I for antigen presentation to CD8 cells. Cell free gp96 associated with antigenic peptides is highly immunogenic. The immune system uses gp96-receptors on ARC and DC as a sophisticated system to detect cell damage and monitor antigenic peptides that are associated with liberated heat shock proteins by engulfing them and cross presenting them to CD8 cells (cross priming). Compared to intact proteins, the association of peptides with gp96 enhances antigen cross priming of CD8 cells between 10,000 to 1 million fold. By replacing the KDEL retention signal of human gp96 with the lgG1-Fc portion we have generated a gp96-fusion protein (gp96-lg) that is secreted from transfected cells. We have shown that gp96-lg secreted from transfected tumor cells in vivo mediated strong, antigen specific CD8-CTL expansion, caused tumor rejection and generated long term anti-tumor immunity. Clinical trials with gp96-lg vaccines in cancer are ongoing. Our data shows that cell secreted gp96-lg triggers recruitment and activation of dendritic cells. DC recruit and activate NK cells and induce a Th1 environment for strong expansion of cognate CD8 CTL stimulated by cross presented, originally gp96-associated peptides engulfed by DC. We also show that cell secreted gp96-Ig upon intraperitoneal immunization induces strong antigen specific CD8 responses in mucosal sites including intraepithelial CD8 cells (IEL). The data indicates that cell secreted gp96-lg induces both, mucosal and systemic CD8-CTL based immunity. Gp96-lg secreted from HIV-antigen expressing cells therefore is expected to provide strong anti HIV immunity systemically and at mucosal sites and provide protection from infection. These hypotheses will be examined in the application. In specific Aim 1, we will examine the route of gp96-immunization in relation to antigen specific IgA and CD8 responses at various systemic and mucosal sites. In addition, polyspecificity of gp96 vaccines will be examined. In specific aim 2, we will study the gp96-vaccine induced CD8 memory response at mucosal and systemic sites and correlate CD8 responses with resistance to viral challenge. The model system will use HLA A2 transgenic mice and HIV expressing vaccinia virus. [unreadable] [unreadable]